Safe Project Design

Our modified strain of Pseudomonas putida KT2442 will be able to overproduce biofilm by using a tight salicylate-controlled expression of signaling (yhjH, pleD*) and structural (lapA, lapC, lapG) genes which control this process. These biofilm-forming bacteria will also be able to consume glycerol by expressing a glycerol transporter (glpF) with the same system, and they are also useful in order to produce many different compounds using that carbon source, such as propionate.Therefore, the main application of our bacteria is the industrial bioremediation and production of compounds.

• Our chassis bacterium, Pseudomonas putida KT2442, belongs to the Risk group 1 and is harmless and non-pathogenic for humans.
• The main risk about the use of a biofilm overproducing bacterium would be the transmission of biofilm production genes to other species, which could lead to the generation of highly resistant bacteria. To avoid this, we do not use plasmid constructs, but our genes are stably inserted in the bacterial genome to avoid horizontal gene transfer, using the miniTn7 tool.
• The miniTn7 tool allows us to avoid many biosafety issues related to plasmid use, such as horizontal gene transfer or antibiotic selection (which can lead to resistance spreading); and it also makes our work much easier and simpler (avoids many problems related to plasmid stability, plasmid copy number and also provides the suitability to multiple bacterial hosts)
• If our project is completely finished and turns into a real product, the whole process can be developed in a biotechnological factory. Within the industrial applications, our modified strain is useful for the generation of products from bioremediation processes. These products might contain other substances originated in the bacterial culture. Therefore, it would be important to purify our compound of interest to avoid the presence of any dangerous substance.
• Since our bacteria is suitable for bioremediation processes, many waste products can be used, therefore any by-product can be easily recycled. Our system does not produce harmful or dangerous wastes.
• Furthermore, we are planning to improve the expression and regulation modules that control our genetic circuit to have an ever tighter regulation. This allows to be able to induce or stop bioremediation and/or production, and the formation or dispersion of biofilm, using molecular switches. This avoids biosafety issues such as the uncontrolled activity of exogenous genes and the harmful effect of an accidental release to the environment.

Safe Lab Work

Since we are working with genetically modified organisms, one risk is the release of these to the environment. The microorganisms we are using are not dangerous for human being, but there is a risk considering the presence of immunocompromised people.

Therefore, the main actions we are taking are the containment of the biological material, the sterilization of the laboratory material, waste and work surfaces, and also to avoid the formation of aerosol particles that could lead to the spread of the bacteria. The use of lab coats and gloves is also mandatory in our lab.

The UPO-Sevilla wet lab team uses the facilities of the Centro Andaluz de Biología del Desarrollo (CABD). This center belongs to the Spanish Public Administration and therefore fulfills all the national safety requirements.

After we started our work, our team instructors and lab technicians provided us safety training. We have been trained about fire drill protocols, handling of chemical products, usage of different laboratory facilities, handling of bacterial cultures, sterilization, different types of waste treatment, everyday-protocols about safety performances. Some of our safety guidelines are:

• Use of labcoats, closed shoes and secluded hair.
• Eating, drinking and smoking in the laboratories is forbidden.
• Use of security glasses, the use of contact lenses is not recommended.
• You must know the location and use of safety showers and fire extinguishers.
• You must read the safety specifications of the products you are going to use, including labels and safety cards.
• Keep the working areas clean and neat.
• Keep the containers for chemical products closed and well organized.
• Avoid the inhalation of chemicals. Use safety gloves and masks and wash your hands often.
• Keep chemical products far from heat and fire sources.
• Close the gas faucet after each use.
• Do not pipet liquids with your mouth.
• Use the fume hood when using volatile chemicals.
• Put the different wastes in the appropriated container.

We also fulfill the two most important requirements in iGEM regarding biosafety: we only work with organisms from Risk Groups 1 or 2, and we completely avoid the release of genetically modified organisms and their products to the wild, by using containment protocols and sterilization of work material. Our team registered two organisms in the check in, our main chassis organism (Pseudomonas putida KT2442) and Pseudomonas mosselii, from which we have obtained a part (epimerase) using gene synthesis technology.

Responsible person for the safety of biology labs at our institution: Isabel Alonso Perez (+34 954977565, CABD).

The laws and regulations that govern biosafety in research laboratories in our region, regarding the use of genetically modified organisms, can be consulted here: http://www.magrama.gob.es/es/calidad-y-evaluacion-ambiental/temas/biotecnologia/organismos-modificados-geneticamente-omg-/legislacion-general/Legislacion_espaniola.aspx